Biosynthetic flexibility of Pseudomonas aeruginosa leads to hydroxylated 2-alkylquinolones with proinflammatory host response

The human pathogen Pseudomonas aeruginosa produces various 4(1H)-quinolones with diverse functions. Among these, 2-nonyl-4(1H)-quinolone (NQ) and its N-oxide (NQNO) belong to the main metabolites. Their biosynthesis involves substrates from the fatty acid metabolism and we hypothesized that oxidized fatty acids could be responsible for a so far undetected class of metabolites. We developed a divergent synthesis strategy for 2′-hydroxy (2′-OH) and 2′-oxo- substituted quinolones and N-oxides and demonstrated for the first time that 2′-OH-NQ and 2′-OH-NQNO but not the corresponding 2′-oxo compounds are naturally produced by PAO1 and PA14 strains of P. aeruginosa. The main metabolite 2′-OH-NQ is produced even in concentrations comparable to NQ. Exogenous availability of β-hydroxydecanoic acid can further increase the production of 2′-OH-NQ. In contrast to NQ, 2′-OH-NQ potently induced the cytokine IL-8 in a human cell line at 100 nм, suggesting a potential role in host immune modulation.


Supplementary Methods a. Materials and Methods
Chemicals and solvents for the synthesis were purchased from Sigma-Aldrich, Carl Roth, Acros Organics or VWR Chemicals and were used without further purification. Distilled technical grade solvents and silica gel 60 A (Carl Roth) were used for silica gel chromatography. Thin layer chromatography (TLC) was performed using aluminium sheets "TLC Silica gel 60 F254" from Merck Millipore® and analysed with short/long wave UV-light or by permanganate staining. NMR spectra were obtained on Bruker Avance-III 400 and Bruker Avance-III 600 NMR spectrometers at ambient temperature. Multiplicities are given as follows: s -singlet, ddoublet, t -triplet, q -quartet, quint. -quintet, m -multiplet. Chemical shifts (δ) are given in parts per million (ppm) relative to the solvent residual signal with CDCl3 δH = 7.26 ppm and δC = 77.16 ppm, DMSO-d6 δH = 2.50 ppm and δC = 39.52 ppm, CD3OD δH = 3.31 ppm and δC = 49.00 ppm. [1] . The obtained data were processed and analysed with MestReNova 14.2.0-26256 software. High-resolution mass spectrometry data were obtained on a Hybrid FT Mass Spectrometer LTQ Orbitrap Velos (Thermo Scientific). LC-MS/MS analysis was performed on Dionex Ultimate 3000 UHPLC (Thermo Fisher Scientific) in combination with Finnigan TSQ Quantum (Thermo Fisher Scientific) and on Vanquish™ Horizon/Flex UHPLC system (Thermo Fisher Scientific) in combination with TSQ Series II Quantum (Thermo Fisher Scientific).

Quantification of quinolones in bacterial cultures
An overnight culture (60 µL) was inoculated into 4 mL of LB medium in a sterile 15 mL polypropylene centrifugal tubes with screw caps (VWR). Caps of the tubes were loosely opened by a 180 degree turn and fixed in this position to ensure equal oxygen supply. Cultures were incubated for 3, 6, 9, 12, and 24 h at 37˚C in a shaking incubator at 180 rpm. After incubation, samples were centrifuged at 4500 rpm for 5 min and supernatants were sterile filtrated. 300 μL of culture supernatant were added in 1.5 mL glass vials (LABSOLUTE, Art. Nr. 7612960) with caps containing a PTFE membrane (LABSOLUTE, Art. Nr. 7623097). 300 μL of EtOAc was added and immediately vortexed for 5 sec. After the separation of organic and water phases, 100 μL of the EtOAc layer was transferred via pipetting into mass spec vials containing a glass insert (MACHEREY-NAGEL, Art. Nr. 702007). The EtOAc was evaporated by a gentle stream of nitrogen. For LC-MS/MS analysis, 100 μL of sample solvent (MeOH/H2O 1:1) was added into glass inserts and the residue redissolved. The experiment was performed in triplicates.

Calibration curves for quantification
MeOH stocks of all calibration standards were prepared at 1 mg/mL in glass vials and stored at -80°C for up to 3 months. Calibration standard samples for quantification were prepared in triplicates by serial dilution in sample solvent (MeOH/H2O 1:1) ( Table S2). Calibration equations and R-values were obtained using the Thermo Xcalibur Quan Browser.

LC-MS/MS analysis
Ultra-high performance liquid chromatography was performed on a Dionex Ultimate 3000 UHPLC (Thermo Fisher Scientific) and Vanquish™ UHPLC system (Thermo Fisher Scientific) using a Nucleodur C18 Gravity-SB 100 x 2 mm, 3 μm column (Macherey-Nagel). The flow rate was 0.5 mL min -1 and the column temperature was held at 40°C. The injection volume was 10 μL. Eluent A was 0.1% formic acid in water and eluent B was 0.1% formic acid in acetonitrile. The gradient was 20-100 % B in 10 min, 100 % B for 2 min, 100-20 % B in 1 min, and 20 % B for 2 min. MS/MS analysis was performed by Finnigan™ TSQ  Quantum (Thermo Scientific) and TSQ  Series II Quantum (Thermo Fisher Scientific) mass spectrometers. A heatedelectrospray ionization (HESI-II probe, Thermo Scientific) was used as an ion source. In the optimized conditions the ion spray voltage was 3500 V, vaporizer temperature 300°C, capillary temperature 380°C, sheath gas pressure 60 psi, ion sweep gas pressure 2 psi, and aux gas 10 psi. The fragmentation pattern of quinolone standards was acquired in a Product Ion Scan mode using a fixed collision energy of 30 eV to fragment the corresponding precursor ion before recording the fragments in a mass range of m/z 130-350. Quinolones we quantified in Selected Reaction Monitoring scan mode. MS/MS spectra were acquired in a positive mode. The software Quan Browser Thermo Xcalibur was used for quantitative analysis. The peak area of the respective product ion was fitted by linear regression versus the known concentrations to generate a standard curve. The LC-MS/MS spectra were made available in the GNPS database under the following spectrum identifiers: CCMSLIB00011427575 (

Methyl 3-oxodecanoate (a)
Compound a was synthesized based on procedures described by Zheng et. al. [2] and Vleeschouwer et. al. [3] . To the solution of methyl 3-hydroxydecanoate (b) (619.0 mg, 3.06 mmol) in DMF (2.5 mL) were added imidazole (417.0 mg, 6.12 mmol) and TBS-Cl (554.0 mg, 3.67 mmol) at room temperature. The mixture was stirred overnight at 0˚C. The resulting reaction mixture was poured into sat. aq. NH4Cl solution and extracted with diethyl ether. The combined organic layers were washed with brine solution and dried over MgSO4. The organic phase was evaporated to dryness to yield product 1a as a colorless oil (m = 851.0 mg, 2.69 mmol, 88 %).
Compound 1a was used in the next step without further purification. Compound 2a was synthesized based on the procedure described by Hodgkinson et. al. [4] . Methyl 3-oxodecanoate (a) (1.39 g, 6.94 mmol) was mixed with para-toluene sulfonic acid (132.0 mg, 694.0 μmol), trimethylorthoformate (1.37 mL, 12.49 mmol), and ethylene glycol (1.55 ml, 27.76 mmol) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 5 h at room temperature under nitrogen atmosphere. 10 mL of 5% aq. Na2HPO4 solution was added to the mixture and allowed to stir for 15 min. 15 mL of diethyl ether was added and the mixture was stirred vigorously for 20 min. Organic and aqueous phases were separated. The organic phase was washed with sat. aq. NaHCO3 solution 3 times, dried over MgSO4, filtered, and concentrated in vacuo. Product 2a was obtained as a slightly yellow oil (m = 1.46 g, 5.98 mmol, 85.9 %) and used in the next step without further purification.

General procedure 1: Generation of aldehydes
Protected methyl ester (1 eq.) was dissolved in dry DCM (4 mL/1 mmol) and cooled to -78°C. DIBAL-H solution (1 M in toluene, 1.15 eq.) was slowly added and the resulting mixture was stirred at -78°C for 2 h. The reaction was quenched by the addition of MeOH (1.5 mL/1 mmol) and sat. Rochelle salt solution (7 mL/1 mmol). The mixture was allowed to reach room temperature. Organic and aqueous layers were separated. The aqueous phase was extracted 2 times with DCM. The combined organic layers were washed with brine solution and dried over MgSO4. The organic phase was evaporated to dryness. The residue was purified by silica column chromatography (petrol ether/EtOAc 3:1) to yield the product.

General procedure 2: Aldol condensation
2-Nitroacetophenone (1.15 eq.) was dissolved in dry DCM (4.9 mL/1 mmol of an aldehyde) and cooled to 0°C. Bu2BOTf solution (1 M in DCM, 2.05 eq.) was added and the mixture stirred for 10 min at 0°C. DIPEA (2.25 eq.) was added as a solution in dry DCM (0.4 mL/1 mmol) and the resulting dark-red mixture was stirred for 30 min at 0°C. The reaction was warmed to room temperature and stirred for 30 min. The mixture was cooled to -78°C and aldehyde (1 eq.) was added slowly as a solution in dry DCM (2.33 mL/1 mmol). The mixture was stirred at -78°C for 30 min, warmed to 0°C, and stirred for 2.5 h at this temperature. The reaction was quenched at 0°C by the addition of pH 7 phosphate buffer (9.3 mL/1 mmol), MeOH (14 mL/1 mmol), and 30% H2O2 (9.3 mL/1 mmol). This mixture was allowed to reach room temperature and stirred vigorously for 5 min. Organic and aqueous phases were separated and the aqueous layer was extracted 2 times with DCM. The combined extracts were washed successively with sat. aq. NH4Cl solution and brine solution. The organic phase was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica column chromatography (petrol ether/EtOAc 2:1) to yield the product.

General procedure 4: Reductive cyclization to form 4-quinolone-N-oxide
Ketone was dissolved in EtOH (42 mL/1 mmol) and 10 % Pt/C catalyst (0.3 mg/1 mg of ketone) was added. The resulting mixture was placed under nitrogen atmosphere and hydrogen gas was supplied from a H2-filled balloon. The mixture was allowed to stir for 3 h at room temperature. The catalyst was removed by filtration and the filtrate was evaporated to dryness. The residue was purified by silica column chromatography to yield the product.

General procedure 5: Reductive cyclization to form 4-quinolone
The ketone was dissolved in EtOH (42 mL/1 mmol) and 10 % Pd/C catalyst (0.3 mg/1 mg of ketone) was added. The resulting mixture was placed under nitrogen atmosphere and hydrogen gas was supplied from a H2-filled balloon. The mixture was allowed to stir for 24 h at room temperature. The reaction mixture was filtered from the catalyst and evaporated to dryness. The residue was purified by silica column chromatography to yield the product.

General procedure 7: Ketal-deprotection
Starting material was dissolved in THF/6 M HCl aq. solution (1:1) (46.5 mL/1 mmol). The resulting mixture was allowed to stir at room temperature for 24 h. The reaction mixture was diluted with distilled water and extracted 2 times with EtOAc. The combined organic layers were washed with sat. aq. NaHCO3 and Brine solutions. The organic phase was dried over MgSO4 and concentrated in vacuo. The residue was purified by prep. RP-HPLC or by silica column chromatography to yield the product.
Note: Signals H-1', C-2, C-3, C-4, C-4a, C-7, C-8, C-8a, C-1', and C-2' couldn't be detected by NMR using different NMR-solvents (MeOH-d4, DMSO-d6, MeCN-d3) at room temperature or 40˚C. Low solubility of the compound in common solvents prevented an adequate concentration for measurements. Methyl ester a (617.0 mg, 3.08 mmol) was dissolved in 4.4 mL of MeOH at +10˚C. 4.4 mL of 0.7 M aqueous NaOH solution (3.08 mmol) was added. The resulting mixture was allowed to stir at +10˚C for 6 h and at room temperature for 18 h. MeOH was evaporated under reduced pressure (≤+30˚C for water bath) and the residue was diluted with cooled water (5˚C). The resulting mixture was acidified to pH 1-2 with 1 M HCl and product 3a precipitated. The product was separated from water by filtration, dried under reduced pressure, and obtained as a white solid (m = 461.7 mg, 2.48 mmol, 80.5 %). Methyl ester b (619.4 mg, 3.06 mmol) was dissolved in 30 mL of MeOH/water (1:1) mixture and NaOH (244.9 mg, 6.12 mmol) was added. The resulting mixture was allowed to stir at room temperature for 24 h. MeOH was evaporated under reduced pressure and the residue was diluted with distilled water. The resulting mixture was acidified to pH 1-2 with 1 M HCl and product 3b was precipitated at 0˚C. The product was separated from water by filtration, dried under reduced pressure, and obtained as a white solid (m = 382.0 mg, 2.03 mmol, 66.3 %).

Sample Integrated Area Sample Integrated Area
Control -       Da production in P. aeruginosa PAO1 in control (orange labels) and after feeding 100 µM deuterated β-hydroxydecanoic acid (3d) to cultures (green labels). The error bars represent the population standard deviation.